We propose to apply the approaches of the organic chemist as well as methods of the enzymologist to probe the nature of hhe active sites of both heart and skeletal muscle creatine kinase, isoenzymes which use creatine, phosphocreatine, ADP and ATP as substrates. These creatine kinase isoenzymes thus serve a dual role as objects for screening creatine, phosphocreatine and nucleotide analogs of potentially great interest in other biological systems. They also serve as prototypal kinases, the mechanism of action none of which is yet well understood. Our general approach involves probing the isoenzymes' active sites with synthetic substrate analogs both to see which functional groups of the substrate are most vital to activity and to see what additional groups may be added without severe detrimental effect on activity. Various physical methods, including kinetic studies and nuclear magnetic resonance studies, are proposed in this probing process.